title: Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8.
abstract: Threo-Ds-3-isopropylmalate dehydrogenase coded by the leuB gene from an extreme thermophile, Thermus thermophilus strain HB8, was expressed in Escherichia coli carrying a recombinant plasmid. The thermostable enzyme thus produced was extracted from the E. coli cells, purified, and crystallized. The enzyme was shown to be a dimer of identical subunits of molecular weight (4.0 +/- 0.5) x 10(4). The Km for threo-Ds-3-isopropylmalate was estimated to be 8.0 x 10(-5) M and that for NAD 6.3 x 10(-4) M. The optimum pH at 75 degrees C in the presence of 1.2 M KCl was around 7.2. The presence of Mg2+ or Mn2+ was essential for the enzyme action. The enzyme was activated about 30-fold by the addition of 1 M KCl or RbCl. The high salt concentration decelerated the thermal unfolding of the enzyme, and accelerated the aggregation of the unfolded protein. Based on these effects, the molecular mechanism of the unusual stability of the enzyme is discussed.
authors.affiliation: Department of Life Science, Tokyo Institute of Technology, Kanagawa.
authors.forename: T
authors.initials: T
authors.lastname: Yamada
authors.forename: N
authors.initials: N
authors.lastname: Akutsu
authors.forename: K
authors.initials: K
authors.lastname: Miyazaki
authors.forename: K
authors.initials: K
authors.lastname: Kakinuma
authors.forename: M
authors.initials: M
authors.lastname: Yoshida
authors.forename: T
authors.initials: T
authors.lastname: Oshima
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D006358:Hot Temperature; D011489:Protein Denaturation; D013379:Substrate Specificity; D013816:Thermodynamics; D013824:Thermus / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D014508:Urea / Q000494:pharmacology
chemical_list: D002412:Cations; D014508:Urea; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase